The fundamental interest of the Laboratory of Myeloid Malignancies is the detection, prevention and treatment of acute myeloid leukemia (AML) relapse. Our work has focused on clinical trials of novel biomarker and immunotherapy approaches, and the development of molecular and genomic laboratory methods to predict development or recurrence of myeloid malignancy. Foundational to our objective has been the development of high sensitivity biomarkers for residual AML in those patients who have been treated to apparent remission but remain at risk of clinical relapse. Previously we have demonstrated the ability to risk stratify AML patients into groups with either high and low leukemic relapse rates, based on pre-transplant a peripheral blood sample, prior to either allogeneic (PMID: 25665046, 27185839) or autologous hematopoietic stem cell transplantation (PMID: 27544285). This year our work in this area has resulted in multiple publications (PMID: 28925935, PMID: 29027628) including contribution to the consensus statement from membership of the European Leukemia Network (ELN) clinical guidelines committee on the use of measurable residual disease (MRD) in AML (PMID: 29330221). In the laboratory our work on AML MRD has focused on the development of a digital targeted RNA-sequencing-based approach, using unique molecular identifiers (UMI), to detect all those newly approved ELN consensus recommendation molecular targets in a single standardized assay. Using iterative laboratory modifications and a novel bioinformatics approach we were able to achieve a greater than one hundred-fold increase in performance compared with commercially available targeted RNA-sequencing approaches. The limit of detection of our new assay is as low as 1 in 100,000 cells which is comparable with quantitative PCR, the current gold standard for AML MRD detection. Our assay, which is customizable and expandable, is the first demonstrated use of high-sensitivity RNA-sequencing for measurable residual disease detection in acute myeloid leukemia and thus could serve as a broadly applicable standardized tool. This work has now been published (PMID: 30171026) and we are currently seeking international cohorts of patient samples for additional validation of clinical utility. For AML MRD in the clinic, we completed analysis of our clinical trial for AML patients with relapsed or refractory disease, PEARL15: Personalized Early Assessment of Response During Salvage Chemotherapy in People with Relapsed or Refractory Acute Myeloid Leukemia (PEARL15, 15-H-0176, NCT02527447). This trial sought to test the hypothesis that high-sensitivity assays for residual disease burden may allow much earlier assessment (day 4 in blood, compared with day 28-42 from cytomorphological examination of bone marrow conventionally) of the success of therapy for relapsed and refractory AML. We have supplemented samples from this NIH trial of relapsed and refractory patients with samples from a cohort of thirty-five newly diagnosed AML patients who underwent initial induction chemotherapy at Duke Cancer Institute. We believe this work represents the first attempt to determine if monitoring of leukemia-associated DNA mutations, or aberrant gene expression, in blood early during cytotoxic therapy can predict clinical response in AML. The work was selected for presentation at the American Society of Clinical Oncology Annual Meeting in 2018 and the full manuscript is currently under review. In 2018 we also completed patient care on our latest clinical trial, PD-AML (17-H-0026, NCT02996474, FDA IND: 131826), which tested the feasibility of a novel combination of pembrolizumab (anti-PD1 immune checkpoint inhibitor) and decitabine (hypomethylating epigenetic modulator) for patients with relapsed/refractory AML. This trial opened in Feb 2017 and was a single arm, open label, investigator sponsored study for ten subjects. Up to 8 cycles of pembrolizumab (every 21 days) were given during the initial induction phase. Ten days of decitabine were given on alternative cycles. Our central hypothesis is that pembrolizumab therapy for refractory and relapsed AML may be associated with changes in the AML clonal composition due to differences in immunogenicity between clones. The oligoclonal nature of AML biology, together with a blood and bone marrow distribution highly amenable to repeated sampling of the sites of disease burden, provides a near unique opportunity to investigate fundamental mechanisms underpinning any treatment efficacy in this new class of immunotherapeutic drugs. The clinical outcomes have been submitted for presentation and additional work on laboratory correlates is currently ongoing. Finally, as a foundation for the analysis of research samples collected on the above trial, we undertook a systematic and comprehensive assessment of human bone marrow using multiple forms of high dimensional single-cell analysis. We recruited a cohort of twenty healthy adult volunteers for collection of research samples from bone marrow aspiration. This group consisted of 10 males and 10 females with ages ranging from 24 to 84 years old. Cryopreserved cells from all twenty donors were analyzed by both droplet-based single cell RNA sequencing and by 13-color flow cytometry using five customized panels designed to allow deep immunophenotyping of the predominant cell populations resident in human bone marrow. Additional cryopreserved vials from eight donors were analyzed by mass cytometry for T cell phenotyping, as well as bulk RNA sequencing. The resulting data set, which includes technical and biological replicates, represents a unique resource of single cell RNA sequencing, flow cytometry and mass cytometry data for a healthy control cohort across the full range of adulthood providing not only cell population frequencies and characteristics, but also highlighting individual variation in human cohorts and reproducibility and correlation between these techniques. A manuscript based on this work is currently under consideration. In summary, the primary interest of the Myeloid Malignancies Section remains the detection, prevention and treatment of AML relapse, in particular the development of molecular and genomic laboratory methods to predict development or recurrence of myeloid malignancy. In the past year we have completed a clinical trial of a novel combination of immunotherapy and hypomethylating therapy for relapsed/refractory AML patients, validated single-cell approaches and created an important reference dataset on human bone marrow, and have developed a new method for detection of AML MRD using high sensitivity targeted RNA sequencing. In the future, we anticipate that the work of the laboratory will continue to focus both on molecular methods for AML MRD detection and the investigation of the human immunology resident within the bone marrow microenvironment, specifically using clinically informative samples at landmark timepoints from patients treated on our immunotherapy trial.